Structure, membrane association, and processing of meprin subunits

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Virginia Tech

Meprins are oligomeric cell-surface metalloproteinases that are expressed at high concentrations in the renal brush border membranes of mice. Meprins consist of two types of subunits, α and β, which are the products of two different genes. The β subunit cDNA was cloned and sequenced from mouse kidneys, and the membrane association and the in vivo proteolytic processing of mouse meprin subunits were investigated. The primary translation product of the meprin β subunit is composed of 704 amino acids, and contains several domains, including a signal sequence at the NH₂-terminus, a prosequence, a protease domain, an adhesion domain (MAM domain), an epidermal growth factor-like domain, a potential transmembrane-spanning domain, and a short cytoplasmic tail. The B subunit is evolutionarily related to the α subunit. The α and β subunit share about 42% overall sequence identity, and have a similar arrangement of functional domains; however, a 56 amino acid segment near the COOH-terminus of a is missing in β, and the signal sequences, transmembrane and cytoplasmic domains share no significant sequence similarity. The protease domains of o and B are 55% identical. NH₂-terminal protein sequencing of detergent-solubilized meprin subunits from mouse kidneys showed that the prosequence in a is removed in the mature subunit. By contrast, only the signal sequence is removed from the mature β subunit NH₂-terminus, and the β subunit retains the prosequence. Further, the mature α subunit, but not the β subunit, is proteolytically processed at the COOH-terminus and does not contain the transmembrane and the EGF-like domains encoded by the meprin α cDNA. The β subunit is a type I integral membrane protein. By contrast, α does not transverse the membrane, and its membrane association depends on disulfide bonds. The oligomeric organization of membrane-bound meprins was analyzed by SDSPAGE under non-reducing conditions, and by isoelectric focusing. ICR mouse kidneys express αβ heterodimers and α₂ homodimers; C3H/He mice contain β₂ dimers. Transfection of COS-1 cells with the full-length meprin α subunit cDNA resulted in the secretion of meprin dimers into the culture medium, indicating that the COOH-terminal transmembrane domain of meprin α subunits is posttranslationally removed from the protein in COS-1 cells, as it is in mouse kidney cells. Replacement of the COOH-terminal 137 amino acids of a with the COOH-terminus of B, or deletion of the 56 amino acid inserted domain in α, resulted in mutant proteins that were not secreted into the medium, but rather were membrane-bound, indicating that the inserted domain of α is essential for proteolytic cleavage and secretion. Deletion of the COOH-terminal 133 residues of a did not affect meprin α dimerization or intracellular transport. The meprin a subunits secreted from transfected COS-1 cells were catalytically inactive, but could be activated by limited proteolysis with trypsin. Thus, processing at the NH,-terminus differed in COS-1 cells and in mouse kidney. COS-1 cells did not remove the prosequence from the α subunit protein, and removal of the prosequence was essential for catalytic activity of the a subunit. These results have implications for the biosynthesis and regulation of a cell surface proteinase, and thus relate to the elucidation of the mechanisms by which biological events at the cell surface are regulated.